Hello Sanjoy Das

Please find the below attached links.

The universal primers 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’) which is given in the following publication 

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0121967

and there are few other link which you can refer for your work

1. https://www.ncbi.nlm.nih.gov/pubmed/12351242 ( same primer used in link 2)

2. http://scialert.net/fulltext/?doi=ajbs.2016.1.9 (same primer used in link 1)

3. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC126540/

hope this information might useful for you.

I’m agree with Kirankumar Nalla. But, if you still face the same manner, you can try this specific primer to amplify your 16S rRNA gene from bacteria (63f and 1387r) based on Marchesi et al.1998.

Somehow, this primer is found to be more efficacy and specifity for bacterial species and environmental samples.

Here is the link:

https://www.ncbi.nlm.nih.gov/m/pubmed/9464425/

Good luck!

Thank you Effendi for agreeing with my answer and recommeding it for solution

You're welcome Kirankumar Nalla!

Dear Kiran Kumar Nalla, Effendi and Enriqueta Garcia,

I thank all of you for your valuable inputs for my query. Previously, I used both 27F and 1492R, but in different combination. I used 27F with 1525R and 8F with 1492R. However, those failed to amplify almost 90% of the isolates. Now as per suggestion of Mr. Nalla and previous literatures, I shall try the combination of 27F and 1492R. I shall also use 63f and 1387r as suggested by Effendi.

My template was pure genomic DNA. So, the presence of lactic acid may not be a problem.

Once again, I thank you all. I shall inform you the outcome.

With regards

Sanjoy Das

Dear Sanjoy Das,

Thank you for your reply. I believe you will obtain good results using universal primer 27f and 1492r. Moreover, if your LAB isolated from aquatic environment, they will suit to specific primer 63f and 1387.

Regards,

Effendi

Effendi, Thank you again

You’re welcome, Sanjoy Das.

27F and 1492R

I think 27 F and 1492 R should work, otherwise, you can also give a try with 533 F and 1492 R whose amplicon size will fit more better for Sanger Sequencing for identifications. In addition, 27 F and 533 R targeting V1-V3 regions can also be another alternative.

Thank you all for your valuable input

Dear Sanjay Das I wish you all the best for your Research

Do not you think the suggestion

27F (5’-AGAGTTTGATCCTGGCTCAG-3’)

1492R (5’-GGTTACCTTGTTACGACTT-3’)

gives you too longer PCR amplicons? Have you got any solid reason?

Thanks

Dear Mehrdad,

I am using 27F and 1492R for last 6 months and getting a good result.

With regards

Sanjoy Das

27F: AGAGTTTGATCMTGGCTCAG

1492R: TACGGYTACCTTGTTACGACTT

Yes. As present, I am using this pair and getting results.